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. 2009 Mar;47(3):807-10.
doi: 10.1128/JCM.01324-08. Epub 2008 Dec 24.

Detection and toxin typing of Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples by PCR

Josephine Wu  1 , Wandi Zhang, Boxun Xie, Maoxin Wu, Xiaodi Tong, Jayant Kalpoe, David Zhang
Affiliations

Detection and toxin typing of Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples by PCR

Josephine Wu et al. J Clin Microbiol. 2009 Mar.

Abstract

Since current microbiology methods are not suitable to detect Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples, we developed a PCR assay to detect toxin-encoding genes and the 16S rRNA gene of C. perfringens. We successfully detected and genotyped C. perfringens in tissue sections from two autopsy cases.

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Figures

FIG. 1.
FIG. 1.
Identification of C. perfringens by macroscopic and microscopic examination. (A) Cut surface of liver (case 2) exhibiting multiple cystic air spaces (“Swiss cheese” appearance) lined by bacteria (left and right insets, magnifications of ×10 and ×40, respectively; both insets stained with hematoxylin and eosin). (B) Section of stomach (case 1) demonstrating colonization of gastric epithelium by numerous boxcar-shaped, gram-positive organisms.
FIG. 2.
FIG. 2.
Detection of 16S rRNA and toxin genes of C. perfringens by PCR. (A) Sensitivity of the PCR assay for the detection of 16S rRNA and toxin genes (cpa, cpb, and etx) of C. perfringens. Genomic DNA was serially diluted 10-fold from 5 × 105 to 5 copies. The results showed that the sensitivity of detecting each gene was between 50 and 500 copies. (B) Specific amplification of the 16S rRNA gene of C. perfringens (279 bp) by PCR. Lane 1, C. perfringens type B (5 × 105 copies); lane 2, C. perfringens type B (5 × 102 copies); lane 3, blank (no target); lane 4, C. difficile; lane 5, C. sordelli; lane 6, P. aeruginosa; lane 7, E. coli; lane 8, S. enterica serovar Typhimurium; lane 9, P. vulgaris. Lanes M in panels A and B contain DNA size markers (pBR322 DNA MspI digest). Genomic DNA (5 × 105 copies) was tested for bacteria other than C. perfringens.
FIG. 3.
FIG. 3.
Genotyping of C. perfringens by PCR. The DNAs extracted from C. perfringens types A to D (ATCC strains) (A) or from tissue sections of case 1 (stomach) and case 2 (liver) (B) were amplified using primers specific for the 16S rRNA gene and each toxin gene (cpa, cpb, and etx) by PCR. Lanes M contain DNA size markers (pBR322 DNA MspI digest).
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References

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